Methods and means for increasing resistance to cell damage

Abstract

Methods are provided to increase resistance to cell damage in a subject. The increase in resistance to cell damage in a subject in the subject is accomplished by decreasing activity of eEF2 kinase in the subject. The eEF2 kinase activity can be decreased by decreasing the amount of functional eEF2 kinase produced by the subject, including contacting the eEF2 kinase with a compound that inhibits phosphorylation of eEF2 kinase substrate or decreasing the amount of functional eEF2 kinase is decreased by reducing expression of a gene encoding the eEF2 kinase.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of International Application No. PCT / US2005 / 022741, filed Jun. 24, 2005, which claims priority to U.S. Provisional Application Ser. No. 60 / 582,411, which was filed on Jun. 24, 2004; and also claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 60 / 819,688, which was filed on Jul. 10, 2006, the disclosures of all three of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of grant R01AG19890 awarded by the U.S. National Institutes of Health (National Institute on Aging).FIELD OF THE INVENTION

[0003] The present invention relates to the field of cell damage and the development of compositions and methods to increase resistance to cell da...

Examples

example 1

Plasmids, Antibodies and Cells

[0128]Retroviral vector used for eEF2 kinase overexpression is constructed by subcloning of eEF2 kinase cDNA from the pSIT retroviral vector7 into LXSN vector (Clontech) using Eco RI / XhoI cloning sites. Stable cell line is prepared through infection of MEFs with pBabe-neo retroviral vector containing SV-40 large T antigen (a kind gift from Dr. J. Yuan). GSE 56 cell lines are established using retroviral vector LXSP, containing GSE5612. LXSP vector is prepared from LXSN vector by the substitution of neomycin marker with puromycin. Antibodies against p21 (F5) and p53 (Ab-1) are from Calbiochem Inc.; antibodies against eEF2 and phospho-eEF2 are from Cell Signaling Inc. eEF2 kinase− / − and eEF2 kinase+ / + primary mouse embryonic fibroblasts used in this study are isolated from 10-12 day embryos following standard protocols. Unless indicated, all cell lines are maintained in DMEM with 10% fetal bovine serum.

example 2

Transfection and Retroviral Infection

[0129]Packaging cells (Phoenix line) are plated in 60-mm plates and transfected with 5 μg of retroviral vector DNA using the standard calcium phosphate procedure. Medium is changed after 8 hours. Virus-containing medium supplemented with 8 μg of Polybrene (Sigma) is collected at 24 and 48 hours post-transfection and used for infection. Infected cells are selected for the resistance to an appropriate selection agent.

example 3

Western Analysis

[0130]For protein expression analysis, cells are washed twice with ice-cold PBS, resuspended in lysis buffer (20 mM Na-phosphate [pH 7.5], 25 mM NaF, 1 mM orthovanadate, 5 mM EDTA), dissolved in Laemmli SDS sample buffer and boiled for 10 minutes. Samples are separated on 5-20% gradient SDS-PAGE and proteins are transferred onto a PVDF membrane. Membranes are incubated with antibodies and developed using ECL Plus reagents (Amersham Biosciences).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of International Application No. PCT / US2005 / 022741, filed Jun. 24, 2005, which claims priority to U.S. Provisional Application Ser. No. 60 / 582,411, which was filed on Jun. 24, 2004; and also claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 60 / 819,688, which was filed on Jul. 10, 2006, the disclosures of all three of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of grant R01AG19890 awarded by the U.S. National Institutes of Health (National Institute on Aging).FIELD OF THE INVENTION

[0003] The present invention relates to the field of cell damage and the development of compositions and methods to increase resistance to cell da...

Examples

example 1

Plasmids, Antibodies and Cells

[0128]Retroviral vector used for eEF2 kinase overexpression is constructed by subcloning of eEF2 kinase cDNA from the pSIT retroviral vector7 into LXSN vector (Clontech) using Eco RI / XhoI cloning sites. Stable cell line is prepared through infection of MEFs with pBabe-neo retroviral vector containing SV-40 large T antigen (a kind gift from Dr. J. Yuan). GSE 56 cell lines are established using retroviral vector LXSP, containing GSE5612. LXSP vector is prepared from LXSN vector by the substitution of neomycin marker with puromycin. Antibodies against p21 (F5) and p53 (Ab-1) are from Calbiochem Inc.; antibodies against eEF2 and phospho-eEF2 are from Cell Signaling Inc. eEF2 kinase− / − and eEF2 kinase+ / + primary mouse embryonic fibroblasts used in this study are isolated from 10-12 day embryos following standard protocols. Unless indicated, all cell lines are maintained in DMEM with 10% fetal bovine serum.

example 2

Transfection and Retroviral Infection

[0129]Packaging cells (Phoenix line) are plated in 60-mm plates and transfected with 5 μg of retroviral vector DNA using the standard calcium phosphate procedure. Medium is changed after 8 hours. Virus-containing medium supplemented with 8 μg of Polybrene (Sigma) is collected at 24 and 48 hours post-transfection and used for infection. Infected cells are selected for the resistance to an appropriate selection agent.

example 3

Western Analysis

[0130]For protein expression analysis, cells are washed twice with ice-cold PBS, resuspended in lysis buffer (20 mM Na-phosphate [pH 7.5], 25 mM NaF, 1 mM orthovanadate, 5 mM EDTA), dissolved in Laemmli SDS sample buffer and boiled for 10 minutes. Samples are separated on 5-20% gradient SDS-PAGE and proteins are transferred onto a PVDF membrane. Membranes are incubated with antibodies and developed using ECL Plus reagents (Amersham Biosciences).

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